A PLATFORM FOR GAG DISACCHARIDE ANALYSIS USING SEC LC/MS

Boston University Medical Campus, Boston MA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The structurally-polydisperse heparan sulfate (HS) chains are responsible for the interactions of HS proteoglycans with a wide variety of proteins, playing critical roles in biology. One HS biosynthesis step, the coordinated N-deacetylation/ N-sulfation on the GlcNAc residues, can be interrupted by the limited supply of 3'-phosphoadenosine 5'-phosphosulfate, and result in the formation of unsubstituted glucosamine residues within HS chains. Although endogenous N-unsubstituted saccharides have only been identified in a handful of studies, they have been implicated to be important in cellular and pathophysiological phenomena. Therefore, there is a clear need to systematically study the occurrence, and determine the structure and distribution patterns of these free-amino-containing HS disaccharides from mammals in a tissue-specific manner. At the present time, all samples received by the MSR for HS analysis are extracted and then digested exhaustively heparin lyases. The disaccharides are then profiled using SEC/MS. The data are used to determine the average chain lengths and average incorporation of sulfate and acetate groups. The data serve to identify the distribution of disaccharides in the internal regions of the parent chain as well as the non-reducing end. It is the non-reducing end that is of particular interest for protein binding studies.